Method for measuring analyte transport across cell membranes using X-ray fluorescence

ABSTRACT

The present invention includes a method and apparatus for measuring the transport of an analyte through a cell membrane. One or more cells expressing a plurality of ion channels are provided. These cells are loaded with the analyte so that the cells contain at least 10 picograms of the analyte within a volume defined by the area of an x-ray excitation beam and a depth of five times the 1/e attenuation depth for at least one characteristic x-ray signal of the analyte as attenuated by water. The unloaded analyte is removed and the amount of the analyte in the cells is measured with x-ray fluorescence.

RELATED APPLICATION/CLAIM OF PRIORITY

This application is a continuation of U.S. application Ser. No.13/871,697 filed Apr. 26, 2013; now U.S. Pat. No. 9,063,154 issued Jun.23, 2015, which is a divisional of U.S. application Ser. No. 12/496,532filed Jul. 1, 2009, now U.S. Pat. No. 8,431,357 issued Mar. 30, 2013;which claims priority to U.S. Provisional Application No. 61/208,115filed Feb. 20, 2009 and U.S. Provisional Application No. 61/133,697filed Jul. 1, 2008; all the foregoing applications and patents areincorporated by reference herein.

GOVERNMENT RIGHTS

This invention was made with US Government support under contract number1R43GM080781-01 awarded by the National Institute of Health. TheGovernment has certain rights to this invention.

FIELD OF THE INVENTION

The present invention relates to a method and apparatus for measuringthe rate of transport through membranes.

BACKGROUND OF THE INVENTION

The identification of potentially important drugs often requires themeasurement of the effect of these drugs on the transport of ionsthrough membranes. For example, some drugs have producedlife-threatening toxicity, associated with a delay in cardiacre-polarization or QT interval prolongation related to ion movement. Forexample, several drugs were recently withdrawn from the market becauseof their effect on the cell membrane transport system.

Ion channels are ubiquitous pore-forming proteins that allow thetransport of ions across cell membranes. Ion channels facilitate themovement of a particular ionic species (for example, Na+,K+, Ca²+, Cr³¹) between cellular compartments and/or across the outer cell membranewith varying ion selectivity. Ion channels are dynamic structures whichrespond to external factors such as voltage gradients, ligands andmechanical forces. The pharmaceutical industry developed therapeuticsthat modify ion channel function. Examples include the anti-epilepticssuch as the sodium channel blocker carbamazepine, the antihypertensivedihydropyridine calcium channel blockers (NORVASC™, which is describedby the trademark owner as a “PHARMACEUTICAL PREPARATION HAVINGANTIANGINAL AND ANTIHYPERTENSIVE PROPERTIES” and sulphonylurea potassiumchannel openers for diabetes (AMARYL™), which is described by thetrademark owner as a “pharmaceutical preparation for use in the oraltreatment of diabetes”. Recent total annual sales of ion channeltargeted drugs are around $20 billion.

Ion channels are difficult molecular targets for drug development.Difficulties arises from the lack of suitable high throughput screeningassay formats, the complexity of ion channel biophysics, and the rangeof potential binding sites and binding modes for drugs.

There remains a need for simpler methods for measuring transport ratesof ions through a membrane.

Therefore, an object of the present invention is to provide a method andapparatus for measuring the transport rates of ions through membranes.

Additional objects, advantages and novel features of the invention willbe set forth in part in the description which follows, and in part willbecome apparent to those skilled in the art upon examination of thefollowing or may be learned by practice of the invention. The objectsand advantages of the invention may be realized and attained by means ofthe instrumentalities and combinations particularly pointed out in theappended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and form a part ofthe specification, illustrate the embodiment(s) of the present inventionand, together with the description, serve to explain the principles ofthe invention. In the drawings:

FIG. 1 shows a flowchart representation of the method of the presentinvention;

FIG. 2 shows another flowchart representation of the method of thepresent invention;

FIG. 3 shows schematic representation of the apparatus of the presentinvention;

FIG. 4 shows another schematic representation of the apparatus of thepresent invention;

FIG. 5 shows a curve of the calculated 1/e attenuation depth for x-raysin water.

FIG. 6 shows a set of rubidium efflux rate data.

FIG. 7 shows a plot of rubidium efflux rates versus inhibitorconcentration.

FIG. 8 shows a schematic of an apparatus of the present invention.

FIG. 9 shows a schematic of an apparatus of the present invention.

FIG. 10 shows a schematic of an apparatus of the present invention.

BRIEF DESCRIPTION OF INVENTION

Briefly, the present invention comprises a method for measuring thetransport of an analyte from a cell. The method comprises the step ofproviding one or more cells which are loaded with an analyte. Theanalyte is then at least partially unloaded from the cells. The analyteis then measured using x-ray fluorescence.

The present invention also comprises a method for measuring thetransport of an analyte into a cell. This method comprises the steps ofproviding one or more cells; increasing the amount of an analyte in thecells; and measuring the analyte using x-ray fluorescence.

The present invention also comprises an apparatus for measuring thetransport of chemicals across cellular barriers. The apparatus comprisesa chamber having an inlet, an outlet, and a means for retaining cellsduring exchange of the solution in the chamber. The chamber also has atleast one location that is translucent to x-rays. The apparatus alsocomprises an X-ray fluorescence spectrometer oriented to analyze thecells through the X-ray translucent location in the chamber.

DETAILED DESCRIPTION

Briefly, the present invention relates to using x-ray fluorescence (XRF)to measure the effective rate of transport of a chemical through amembrane.

An embodiment of the method of the present invention is shown in FIG. 1.This embodiment comprises the steps of providing cells which are loadedwith an analyte. The cells are then exposed to conditions that causethem to unload the analyte. The analyte is measured with x-rayfluorescence. Preferably, the unloaded analyte is substantially removedfrom the volume defined by the area of the x-ray fluoresceneceexcitation beam that is incident on the cells and a depth of five timesthe 1/e depth for at least one characteristic signal of the analyte inwater.

An alternative embodiment of the method of the present invention isshown in FIG. 2. This embodiment comprises the steps of providing cells,which are then loaded with the analyte, and measuring the analyte usingx-ray fluorescence. Preferably, before the cells are loaded, the analyteis substantially depleted in the volume defined by the area of the x-rayfluorescenece excitation beam that is incident on the cells and a depthof five times the 1/e depth for at least one characteristic signal ofthe analyte in water.

In both the embodiments of the method of the present invention, thecharacteristics of the similar components and steps are the same.

The cells are preferably living, biological cells, which preferablyexpress ion channels. It should be understood that cells may be part ofa tissue; or that the term cell may refer to subcellular components withmembranes, such as mitochondria; or other subcellular components thatprovide restricted analyte transport such as endoplasmic reticulum, ormicelles or similar. More preferably, the cells overexpress ionchannels. Generally, the cells should be substantially bounded by aphysical barrier, such as a membrane or a wall, which encloses a volumeof material, such as the analyte, water, proteins, DNA and otherbiological chemicals. The physical barrier has the characteristic thatit may pass the analyte at different rates when it is exposed todifferent stimuli. For example, the physical barrier may be a cellmembrane comprising an ion channel or other membrane protein, where theion channel passes ions at different rates in the presence of inhibitorsor activators. Preferred cells are adherent. An example of a cell linethat is compatible with the present invention is the hERG CHO-K1 cellline, available from CreaCell, Biopolis, 5, avenue du Grand Sablon,38700 La Tronche, France.

The analyte preferably comprises a chemical element having an atomicnumber greater than 10, and more preferably a chemical element selectedfrom the list of zinc, cadmium, thallium, sodium, potassium, rubidium,cesium, magnesium, calcium, barium, strontium, chlorine, bromine, andiodine. More preferably, the analyte comprises a chemical element thathas at least one characteristic x-ray fluorescence emission signalhaving an energy of 2.5 KeV or greater. The cells preferably incorporatean amount of the analyte such that a population of cells within a volumedefined by the area of the x-ray excitation beam from the x-rayfluorescence instrument that is incident on the sample and a depth offive times the 1/e attenuation depth for at least one characteristicx-ray signal of the analyte as attenuated by water contains at least 10picograms of the analyte; the 1/e depths for x-ray energies between 1KeV and 20 KeV are shown in FIG. 5. More preferably the cells comprise,incorporate or internalize an amount of the analyte such that apopulation of cells within a volume defined by the area of the x-rayexcitation beam from the x-ray fluorescence instrument that is incidenton the sample and a depth of five times the 1/e attenuation depthcontains between 10 nanomolar and 5 molar concentration of the analyte.

The cells are preferably immobilized; immobilized in this context meansthat a quantity of cells equivalent to at least 1% of the cells whichare in the beam path of x-ray fluorescence excitation beam at thebeginning of an x-ray fluorescence measurement are retained in the beampath of x-ray fluorescence excitation beam for a period of time which isgreater than the measurement time of the x-ray fluorescence measurement.More preferably, immobilized in this context means that a quantity ofcells equivalent to at least 1% of the cells which are in the beam pathof x-ray fluorescence excitation beam at the beginning of an x-rayfluorescence measurement are retained in the beam path of x-rayfluorescence excitation beam for at least 10 seconds.

Examples of methods to immobilize the cells include the following: thecells may be immobilized by means of a filter that retains the cells andallows the first solution and second solution to pass. Preferably thecells may be immobilized by adherence to a solid support, such as foam,sheet, film, membrane, scaffold, gel, adherence factor, adherent cellline, tissue, differential diffusion rates, fluidic forces, ordifferentiated cell or other surface on which the cells can adhere. Thecells may be adherent or be part of a tissue or other differentiatedcell mass. If a foam is used, an open cell foam is preferable, and apartially reticulated open cell foam is most preferable.

The analyte may be conveniently unloaded by removing any solution inwhich the cells are supported, and adding a second solution. The secondsolution may be added after removing the first solution, for example, bypouring out the first solution or filtering the cells from the firstsolution, followed by adding the second solution. The second solutionmay alternatively be added to the first solution, so that the secondsolution displaces the first solution. The second solution is aconvenient method to add one or more stimuli to release the analyte. Thestimuli preferably comprise at least one of the materials selected fromthe list of a chemical that induces the analyte to traverse the physicalbarrier, a chemical that inhibits the analyte from traversing thephysical barrier, a solvent that is substantially depleted in theanalyte, a chemical that induces ion channel activity, and a chemicalthat inhibits ion channel activity; this inhibition of ion channelactivity may be direct, for example, the inhibitor binds to the ionchannel; or indirectly, for example the chemical inhibits ion channelactivity by binding to a helper protein or cofactor or similar; themechanism of the inhibition is not important for the functioning of thepresent invention. The first solution may conveniently be removed bydisplacing or diluting the first solution with the second solution. Thefirst solution may also be removed by draining, decanting, or otherwisephysically moving a substantial portion of the first solution with orwithout simultaneously replacing it with a second solution. If the cellsare exposed to a second solution, the second solution preferably has adifferent composition than the first solution. The second solutionpreferably comprises one or more of the following chemical(s): achemical to replace the analyte, such as replacing rubidium ions withpotassium ions; a chemical to induce channel activity, such as a highconcentration of potassium; and a chemical to modify ion channelactivity, such as astemizole, cisapride, or terfenadine. Examples ofthis difference in composition may be the identity or concentration ofone or more solutes, or the identity of the solvent, including mixturesof solvents. Preferably the difference between the first solution andthe second solution is that the second solution is substantiallydepleted in the analyte. In this context, “substantially depleted” meansthat the concentration of the analyte in the second solution is at leastten times less than the concentration of the analyte in the cells whenthe cells are first exposed to the second solution. More preferably,“substantially depleted” means that the concentration of the analyte inthe second solution is at least one hundred times less than theconcentration of the analyte in the cells when the cells are firstplaced in the second solution.

The cells are analyzed by x-ray fluorescence. Examples of x-rayfluorescence spectrometers that may be conveniently used with thepresent invention are the EDAX Eagle XPL energy dispersive X-rayfluorescence spectrometer, equipped with a microfocus X-ray tube, apolycapillary x-ray focusing optic, lithium drifted silicon solid-statedetector, processing electronics, and vendor supplied operatingsoftware; and the KEVEX™ Omicron model 952-102 with a collimated Xraytube and a Si(Li) detector, processing electronics, and vendor suppliedoperating software. The mark KEVEX™ is described by its trademark owneras “laboratory equipment; namely, electron microscopes, x-rayspectrometers, spectral analysis systems, for use in the biomedical,industrial and educational fields, for elemental analysis and digitalimaging”. The x-ray fluorescence spectrometer preferably comprises amoveable stage, and more preferably a stage that may be moved in atleast two dimensions, and most preferably a stage that may be moved inat least three dimensions. Preferred x-ray sources emit polychromaticx-rays, or for which the measured spectrum of the x-ray tube or themeasured spectrum of scattered x-rays from a hydrocarbon samplecomprises x-rays having at least two different energies separated by atleast 0.5 KeV.

The analyte is preferably measured while it is co-located with the cellsbecause it allows real time or near real time measurements and easilyanalyzed analyte efflux measurement, but the analyte may be measuredafter the cells are lysed, after the analyte has been unloaded from thecells, or the difference between the analyte which is loaded in thecells and the amount of analyte to which the cells have been exposed maybe measured. Removing or reducing the matrix, such as by lysing thecells or drying the cells can produce superior measurement limits.

This embodiment of the method of the present invention may readily bemultiplexed.

Multiple measurements may be obtained over a period of time or withdifferent first solutions or second solutions. This allows kineticparameters to be calculated and inhibition constants such as an IC₅₀ tobe calculated.

An embodiment of the apparatus of the present invention is shown in FIG.3. Apparatus 2 comprises X-Ray Fluorescence Spectrometer 4, which isoriented to analyze Cells 12 in Chamber 6. Chamber 6 comprises an Inlet8 and an Outlet 10. Chamber 6 also comprises either a filter to retainCells 12 or a surface upon which cells can adhere or another means toretain Cells 12; if a foam is used, the foam is preferably an open cellor partially reticulated foam; preferably, the means of retaining Cells12 is sufficient to retain Cells 12 during an exchange of a solutionthat substantially surrounds Cells 12. The means for retaining Cells 12is preferably one or more surfaces to which an adherence cell mayadhere. At least one portion of Chamber 6 must be translucent ortransparent to x-rays. Chamber 6 may be a discrete unit, or it may be aportion of a conduit which retains cells.

Cells 12 should have a physical barrier, such as a membrane or a wall,which substantially encloses a volume of material, such as an analyte,water, proteins, DNA and other biological chemicals. The physicalbarrier has the characteristic that it may pass the analyte at differentrates when it is exposed to different stimuli. For example, the physicalbarrier may be a cell membrane with a plurality of ion channels, wherethe plurality of ion channels pass ions at different rates in thepresence of inhibitors or activators. Preferred cells are adherent.

X-Ray Fluorescence Spectrometer 4 comprises an x-ray excitation sourceand an x-ray detector. Examples of x-ray fluorescence spectrometers thatmay be used with the present invention are the EDAX Eagle XPL energydispersive X-ray fluorescence spectrometer, equipped with a microfocusX-ray tube, a polycapillary x-ray focusing optic, lithium driftedsilicon solid-state detector, processing electronics, and vendorsupplied operating software; and the KEVEX™ Omicron model 952-102 with acollimated X-ray tube and a Si(Li) detector, processing electronics, andvendor supplied operating software. The x-ray fluorescence spectrometerpreferably comprises a moveable stage, and more preferably a stage thatmay be moved in at least two dimensions, and most preferably a stagethat may be moved in at least three dimensions. Preferred x-ray sourcesemit polychromatic x-rays, or for which the measured spectrum of thex-ray tube or the measured spectrum of scattered x-rays from ahydrocarbon sample comprises x-rays having at least two differentenergies separated by at least 0.5 KeV.

The portion of Chamber 6 that is translucent or transparent to x-rayshas the characteristic that the X-ray translucent location passes atleast 0.1% of the highest energy x-ray fluorescence signal that areemitted by the portion of the analyte that is located within 1 micron ofthe x-ray translucent location and that are normal to the x-raytranslucent location and incident upon the x-ray translucent location.

Chamber 6 preferably does not leach the analyte, and more preferablydoes not comprise the analyte, and most preferably does not comprise thesame element as the element in the analyte that is being measured usingx-ray fluorescence. Chamber 6 is preferably biologically compatible, sothat at least the surfaces of Chamber 6 that contact the first solutionor second solution are not toxic to cells; in this context, not toxic tocells means that the surface of Chamber 6 does not kill more than 50% ofthe cells within 30 minutes. Chamber 6 also optionally allows cells toadhere to at least one of its inner surfaces; this inner surface may bea foam inside Chamber 6.

The means for retaining Cells 12 may be either a filter (i.e. acomponent that retains the cells and allows the solution to pass) or asurface to which the cells may adhere. An example of a filter is aregenerated cellulose filter. Preferably, the means for retaining Cells12 is a surface to which adherent cells may adhere. If a surface towhich cells may adhere is used; examples of surfaces are polystyrene,polycarbonate, and polyurethane; the examples of the surface are sheets,films foams, and other shapes. The surfaces are optionally treated withchemicals to promote cell adherence, for example, by treatment withcollagen-l or poly-l-lysine or etching. The means for retaining Cells 12preferably is disposed such that the Cells 12 which are in the volumedefined by the area of the x-ray excitation beam from the x-rayfluorescence instrument that is incident on the sample and a depth offive times the 1/e attenuation depth for at least one characteristicx-ray signal of the analyte as attenuated by water contains at least 10picograms of the analyte; the 1/e depths for x-ray energies between 1KeV and 20 KeV are shown in FIG. 5. More preferably the means forretaining Cells 12 is disposed such that the Cells 12 which are in thevolume defined by the area of the x-ray excitation beam from the x-rayfluorescence instrument that is incident on the sample and a depth offive times the 1/e attenuation depth for at least one characteristicx-ray signal of the analyte as attenuated by water contains between 10nanomolar and 5 molar concentration of the analyte.

Optionally, and preferably, Apparatus 2 further comprises flow controlsto modify a solution entering the chamber. This flow control couldcomprise a pump which is capably of providing a solution with a gradientof concentrations of different solutes or solvents.

This embodiment of the apparatus of the present invention may readily bemultiplexed, for example by etching or engraving multiple copies ofApparatus 2 in a single block of plastic or by attaching multipleindividual Apparatuses 2 together.

Another embodiment of the apparatus of the present invention is shown inFIG. 4. Apparatus 102 comprises X-Ray Fluorescence Spectrometer 104disposed to analyze cells in Chamber 106; although X-Ray FluorescenceSpectrometer 104 is shown measuring the cells through an opening inChamber 106, it should be understood that X-Ray FluorescenceSpectrometer 104 may be disposed in any direction that allows it tomeasure the cells, e.g. measuring the cells through the bottom surfaceof Chamber 106 is acceptable if that surface is translucent to x-rays.Chamber 106 also optionally comprises either a filter to retain theCells 112 and/or a surface upon which cells can adhere, shownschematically as Cell Retainer 114. Cell Retainer 114 may be a foam, astructured surface, a gel, tissue sample, or other means for retainingCells 112. Alternatively, the cells may be allowed to settle e.g. usinggravity or centrifugation or reduced pressure through a filter, or othermeans for separating the cells from the solution. At least one portionof Chamber 106 must be translucent or transparent to x-rays; thistranslucent or transparent portion of Chamber 106 may be an opening inChamber 106. Chamber 106 may be a discrete unit, or it may be part of amultiplexed unit containing multiple copies of Chamber 106.

The orientation of Chamber 106 and the x-ray excitation beam and thex-ray detector must allow at least a portion of the population of cellsto occupy the volume defined by the intersection of the x-ray excitationbeam path and the viewable volume of the x-ray detector. Preferably theorientation of Chamber 106 and the x-ray excitation beam and the x-raydetector must allow at least a portion of the population of cellscomprising at least 100 picograms of the analyte to occupy the volumedefined by the intersection of the x-ray excitation beam path and theviewable volume of the x-ray detector. More preferably the orientationof Chamber 106 and the x-ray excitation beam and the x-ray detector mustallow at least a portion of the population of cells comprising at least100 picograms of the analyte to occupy the volume defined by theintersection of the x-ray excitation beam path and the viewable volumeof the x-ray detector, and any barrier between the cells and the x-rayfluorescence detector attenuates the portion of the highest energy x-rayfluorescence signal and that is emitted normal to the barrier by theanalyte that is located within 5 microns of the barrier by less thanabout 99.9% or allows at least 0.1% of the highest energy x-rayfluorescence signal from analyte to pass.

X-Ray Fluorescence Spectrometer 104 comprises an x-ray excitation sourceand an x-ray detector. Examples of x-ray fluorescence spectrometers thatmay be used with the present invention are the EDAX Eagle XPL energydispersive X-ray fluorescence spectrometer, equipped with a microfocusX-ray tube, a polycapillary x-ray focusing optic, lithium driftedsilicon solid-state detector, processing electronics, and vendorsupplied operating software; and the KEVEX™ Omicron model 952-102 with acollimated X-ray tube and a Si(Li) detector, processing electronics, andvendor supplied operating software. The x-ray fluorescence spectrometerpreferably comprises a moveable stage, and more preferably a stage thatmay be moved in at least two dimensions, and most preferably a stagethat may be moved in at least three dimensions. Preferred x-ray sourcesemit polychromatic x-rays, or for which the measured spectrum of thex-ray tube or the measured spectrum of scattered x-rays from ahydrocarbon sample comprises x-rays having at least two differentenergies separated by at least 0.5 KeV.

This embodiment of the apparatus of the present invention may readily bemultiplexed, for example by etching or engraving multiple copies ofApparatus 102 in a single block of plastic or by attaching multipleindividual Apparatuses 102 together.

EXAMPLE 1

Human-ether-a-go-go expressing Chinese Hamster Ovary Cells (hERG CHO-K1)obtained from CreaCell, Biopolis 5, Avenue du Grand Sablon, 38700 LaTronche, FRANCE, were grown in T150 flasks at 37° C. under 5% carbondioxide in growth media consisting of F-12 Nutrient mixture (HAM,obtained from Invitrogen, part number 21765-029), 10% Foetal BovineSerum (FBS), antibiotic-antimycotic (10,000 units penicillin, 10,000 ugstreptomycin, 25 ug amphotericin B/mL, obtained from Invitrogen, partnumber 15240-062), and 1.2 mg/ml geneticin (obtained from Invitrogen,part number 10131-027). When cells reached approximately 80% confluency,the cells were trypsinized (using Trypsin-EDTA) and counted (using ahemacytometer). Cells were resuspended in growth media at aconcentration of 0.1×10⁶ cells in 300 microliters of growth media. 300microliters of cell suspension were transferred to polyurethane foam(PUF) (30 ppi, partially reticulated, from McMaster-Carr part number86225K11 cut into one-quarter inch diameter discs) pretreated with 10micrograms type 1 collagen from calf skin (obtained from Sigma, partnumber C8919) per cm² of PUF in a 48-well plate. Individual PUFs wereincubated at 37° C. at 5% carbon dioxide for approximately 24 hours atwhich time they were transferred to a 60 millimeter diameter petri dishand covered with growth media and incubated at 37° C. at 5% carbondioxide until approximately 80% confluent. Individual PUFs were thenrinsed three times in Rb-/K-buffer consisting of 0.22 um filtered, pH7.4 aqueous buffer containing 155.4 mM sodium chloride, 2 mM calciumchloride, 0.8 mM sodium dihydrogen phosphate, 1 mM magnesium chloride, 5mM glucose, and 25 mM HEPES buffer.

PUFs were loaded with Rb ions via a three hour incubation at 37° C. at5% carbon dioxide in 8 mL Rb+ Loading Buffer consisting of 0.22 umfiltered, pH 7.4 aqueous buffer containing 5.4 mM rubidium chloride, 150mM sodium chloride, 2 mM calcium chloride, 0.8 mM sodium dihydrogenphosphate, 1 mM magnesium chloride, 5 mM glucose, and 25 mM HEPESbuffer. PUFs were treated with channel inhibitors by incubating invarying concentration terfenadine in dimethyl sulfoxide (DMSO) for 30minutes at 37° C. at 5% carbon dioxide, as shown in FIG. 6. IndividualPUFs were then rinsed three times in Rb-/K-buffer consisting of 0.22 umfiltered, pH 7.4 aqueous buffer containing 155.4 mM sodium chloride, 2mM calcium chloride, 0.8 mM sodium dihydrogen phosphate, 1 mM magnesiumchloride, 5 mM glucose, and 25 mM HEPES buffer.

Three individual PUFs were then inverted and stacked in Apparatus 202 asshown in FIG. 8. FIG. 8 shows Apparatus 202, which comprises Chamber206, Inlet 208 that allows liquid to enter Chamber 206, and Outlet 210which allows liquid to exit from Chamber 206. Cell Loaded Foam 204comprised set of three foam PUFs loaded with CHO-KI cells loaded withrubidium as described above. Cell Loaded Foam 204 was placed in Chamber206. A sheet of ULTRALENE™(XRay Translucent Window 212) and an o-ring(Retaining Ring 214) were used to seal Chamber 206. The mark ULTRALENE™is described by its trademark owner as “thin polymeric film forlaboratory use as cell windows for spectroscopic analysis”. FIG. 8 doesnot show the x-ray fluorescent spectrometer or the fluid control system.FIG. 9 shows a schematic of Apparatus 202, excluding the Cell LoadedFoam 204, the X-Ray Translucent Window 212, and the Retaining Ring 214.FIG. 10 shows a schematic of Apparatus 302, which comprises Apparatus202. Apparatus 302 comprises X-Ray Tube 302 and X-Ray Detector 304,which are disposed to analyze the cells in Apparatus 202. Apparatus 302also shows the inlet fluidics, comprising Inlet Fitting 306 whichattaches Inlet Tube 308 to Apparatus 202, and Pump 310 which pumpsliquid from Inlet Reservoir 312 through Inlet Tube 308 to Apparatus 202.Apparatus 302 also comprises outlet fluidics, comprising Outlet Fitting314 which connects Apparatus 202 to Outlet Tube 316. Outlet Tube 316allows liquid to exit Apparatus 202 and reach Outlet Reservoir 318.

The inlet line was then purged with 50 mM K Flow Buffer consisting of0.22 um filtered, pH 7.4 aqueous buffer containing 50 mM potassiumchloride, 150 mM sodium chloride, 2 mM calcium chloride, 0.8 mM sodiumdihydrogen phosphate, 1 mM magnesium chloride, 5 mM glucose, and 25 mMHEPES buffer. The inlet line was then connected to the flow deviceinlet. A static x-ray fluorescence reading was taken using an x-rayfluorescence spectrometer equipped with a Rh x-ray tube run at 25 kV and0.1 mA, lithium-drifted silicon detector, 1 mm collimator; after whichthe flow buffer pump was started. Subsequent x-ray fluorescence readingswere taken under flow conditions over a period of time. Then entire setof flow data for varying inhibitor concentrations was then compiled, andplotted as shown in FIG. 6. The rates of rubidium efflux were found byfitting each set of efflux data shown in FIG. 6. The efflux rates, whichdescribe the loss of rubidium signal, were then plotted versus theconcentration of added terfenadine, to obtain an IC50, as shown in FIG.7.

The embodiment(s) were chosen and described in order to best explain theprinciples of the invention and its practical application to therebyenable others skilled in the art to best utilize the invention invarious embodiments and with various modifications as are suited to theparticular use contemplated. It is intended that the scope of theinvention be defined by the claims appended hereto.

What is claimed is:
 1. A method for measuring the transport of ananalyte through a cell membrane, comprising the steps of providing oneor more cells expressing a plurality of ion channels, loading the one ormore cells with an analyte so that the cells comprise at least 10picograms of the analyte within a volume defined by the area of an x-rayexcitation beam and a depth of five times the 1/e attenuation depth forat least one characteristic x-ray signal of the analyte as attenuated bywater, removing the unloaded analyte substantially from the volume,exciting the analyte with a polychromatic x-ray beam, and measuring thex-ray fluorescence of the analyte with an x-ray detector.
 2. The methodof claim 1, further comprising unloading the cells and measuring theanalyte after it is unloaded from the cells.
 3. The method of claim 1,further comprising lysing the cells prior to measuring the x-rayfluorescence.
 4. The method of claim 1, further comprising reducingmatrix of the cells prior to measuring the x-ray fluorescence.
 5. Themethod of claim 1, further comprising drying the cells prior tomeasuring the x-ray fluorescence.
 6. The method of claim 1, whereinprior to loading the cells with the analyte, the analyte issubstantially depleted in the volume defined by the area of the x-rayfluorescence excitation beam that is incident on the cells and a depthof five times the lie depth for at least one characteristic signal ofthe analyte in water.
 7. The method of claim 1, wherein the analytecomprises an element having an atomic number greater than
 10. 8. Themethod of claim 1, wherein the analyte comprises a chemical elementselected from the group consisting of zinc, cadmium, thallium, sodium,potassium, rubidium, cesium, magnesium, calcium, barium, strontium,chlorine, bromine, and iodine.
 9. The method of claim 1, wherein thecells are retained in a chamber and wherein at least one portion of thechamber is translucent or transparent to x-rays.
 10. The method of claim9, wherein the portion of the chamber that is translucent to x-rayspasses at least 0.1% of the highest energy x-ray fluorescence signalthat are emitted by the portion of the analyte that is located within 1micron of the x-ray translucent location and that are normal to thex-ray translucent location and incident upon the x-ray translucentlocation.
 11. The method of claim 1, wherein the cells are immobilizedsuch that a quantity of cells equivalent to at least 1% of the cellswhich are in the beam path of x-ray fluorescence excitation beam at thebeginning of an x-ray fluorescence measurement are retained in the beampath of x-ray fluorescence excitation beam for a period of time which isgreater than the measurement time of the x-ray fluorescence measurement.12. The method of claim 1, wherein the cells are immobilized such that aquantity of cells equivalent to at least 1% of the cells which are inthe beam path of x-ray fluorescence excitation beam at the beginning ofan x-ray fluorescence measurement are retained in the beam path of x-rayfluorescence excitation beam for at least 10 seconds.
 13. The method ofclaim 1, wherein the cells are retained in a chamber, and theorientation of the chamber and the x-ray excitation beam and the x-raydetector allows at least a portion of the population of cells to occupythe volume defined by the intersection of the x-ray excitation beam pathand the viewable volume of the x-ray detector.
 14. The method of claim13, wherein the portion of the population of cells comprises at least100 picograms of the analyte occupying the volume defined by theintersection of the x-ray excitation beam path and the viewable volumeof the x-ray detector.
 15. The method of claim 14, wherein the portionof the population of cells comprising at least 100 picograms of theanalyte to occupy the volume defined by the intersection of the x-rayexcitation beam path and the viewable volume of the x-ray detector, anybarrier between the cells and the x-ray fluorescence detector attenuatesthe portion of the highest energy x-ray fluorescence signal and that isemitted normal to the barrier by the analyte that is located within 5microns of the barrier by less than about 99.9% or allows at least 0.1%of the highest energy x-ray fluorescence signal from analyte to pass.